Antibodies specific to human T-cell immunoglobulin and ITIM domain (TIGIT)

ABSTRACT

Monoclonal antibodies that recognize TIGIT and inhibit its suppressive activity on T-cells are provided as well as pharmaceutical compositions including them and methods for their use in cancer immunotherapy and in diagnosis and treatment of immune disorders.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Mar. 1,2018, named “SequenceListing.txt”, created on Feb. 26, 2018 (8.67 KB),is incorporated herein by reference.

FIELD OF THE INVENTION

The invention is in the field of immunotherapy and relates to monoclonalantibodies and fragments thereof which bind to the protein human TIGIT,to polynucleotide sequences encoding these antibodies and to hybridomacells producing these antibodies. The invention further relates totherapeutic and diagnostic compositions comprising these antibodies andto methods of treating and diagnosing diseases, particularly cancer,using these monoclonal antibodies.

BACKGROUND OF THE INVENTION

Cancer immunotherapy is utilized for generating and augmenting ananti-tumor immune response, e.g., by treatment with antibodies specificto antigens on tumor cells, with cancer cells fused with antigenpresenting cells (APC), or by specific activation of anti-tumor T cells.

Natural killer (NK) cells of the innate immune system play an importantrole in immune surveillance of tumors (Smyth et al, Nat Immunol. 2001.April; 2(4):293-9). NK cells kill MHC class I-deficient cells, tumors,viruses, parasites and bacteria directly and indirectly. NK cellactivity is controlled by a balance of signals, delivered by inhibitoryand activating NK cell receptors. There are several activating NK cellreceptors that recognize various ligands, which can be stress-induced,self-molecules, viral components or tumor proteins (Koch et al, Trendsin immunology 2013, 34, 182-191; Seidel et al., Cellular and molecularlife sciences, 2012). However, the exact mechanisms by which NK cellsrecognize and eliminate tumors via activating receptors are notwell-understood, in part, because the tumor ligands of severalactivating NK cell receptors are unknown. In contrast, the identity ofthe ligands recognized by inhibitory NK cell receptors is well defined.NK cells express a vast repertoire of inhibitory receptors (Gardiner C.M., International journal of immunogenetics 2008, 35, 1-8; Gonen-Grosset al, PloS one 2010, 5, e8941; Lankry et al., Methods in molecularbiology 2010, 612, 249-273). Most of these inhibitory receptors belongto the KIR (Killer Inhibitory Receptors) family, recognizing bothclassical and non-classical major histocompatibility complex (MHC) classI proteins. KIRs are stochastically expressed on the NK cell surface,thus NK cells in a given individual express selected KIRs. NK cells alsoexpress additional inhibitory receptors that do not recognize MHC classI proteins, such as CEACAM1, CD300a and TIGIT (T-cell Immunoglobulin andITIM Domain).

The human TIGIT protein is expressed on all NK cells, as well as onother immune cells such as T regulatory (Treg) CD8+ cells and Tumorinfiltrating lymphocytes (Stanietsky et al., PNAS. 2009, 106,17858-17863). It recognizes two very well defined ligands: poliovirusreceptor (PVR, CD155) and Nectin2 (PVRL2/CD112) that are expressed onnormal epithelia as well as over expressed on various tumor cells. Therecognition of these ligands leads to the delivery of an inhibitorysignal mediated by two motifs present in the cytoplasmatic tail ofTIGIT: the immunoreceptor tail tyrosine (ITT)-like and theimmunodominant tyrosine based inhibitory (ITIM) motifs (Liu et al., Celldeath and differentiation 2013, 20, 456-464; Stanietsky et al., Europeanjournal of immunology, 2013. 43, 2138-2150). TIGIT, through its ITIMdomain, inhibit NK cytotoxicity leading to immune evasion mechanism oftumor cells.

TIGIT expression on NK cells also serves as the receptor that binds theFap2 protein of the anaerobic Gram-negative bacterium Fusobacteriumnucleatum (F. nucleatum). The interaction between F. Nucleatum and TIGITleads to reduced NK cytotoxic activity. Fusobacteria are often enrichedin patients with intestinal inflammation and cancer. It was suggestedthat F. nucleatum binding to TIGIT facilitates tumor evasion from NKassociated cytotoxicity (Gur et al., Immunity. 2015 Feb. 17; 42(2):344-355), providing an explanation on how bacteria found within tumors,in particular, F. nucleatum, promote tumor proliferation and enhancetumor progression (Jobin, Cancer discovery. 2013; 3:384-387; Sears andGarrett, Cell Host Microbe. 2014; 15:317-328).

Recently, it was shown that TIGIT and PD-1 impair tumor antigen-specificCD8+ T cells in melanoma patients (Chauvin el al. J Clin Invest. 2015;125(5):2046-2058). In addition, TIGIT expression by CD8+tumor-infiltrating lymphocytes (TILs) has been reported using geneexpression analyses in a number of mouse and human solid tumorsincluding lung, colon, breast, uterine, and renal cancers. ElevatedTIGIT expression appears to correlate with CD8 and PD-1 expression.TIGIT expression on CD8+ TILs was observed in mouse tumors and in 3human tumor samples, including non-small-cell lung and colon cancers(Johnston R J, et al. Cancer Cell. 2014; 26(6):923-937).

It was suggested that TIGIT contributes to functional T-cell impairmentand associates with poor clinical outcome in acute myeloid leukemia(AML). A study recently published by Kong et al. (Clin Cancer Res;22(12); 3057-66), suggests that blockade of TIGIT to restore T-cellfunction and antitumor immunity may represent a novel effective leukemiatherapeutic.

WO 2004/024068 describes agonists and antagonists to the moleculePRO52254, later identified as TIGIT, for treatment of autoimmunediseases and cancer without disclosing actual antibodies.

WO 2006/124667 discloses modulation of the protein zB7R1 (TIGIT) bymonoclonal antibodies that block TIGIT binding to its ligand PVR. Nobinding affinities are provided.

WO 2009/126688 discloses TIGIT, and its ligand PVR, as targets formodulation of immune responses and suggests agonists and antagonists ofthese proteins for diagnosis and treatment of immune-related andinflammatory diseases.

WO 2015/009856 discloses combinations of programmed death 1 polypeptide(PD-1) antagonists and anti TIGIT antibodies for treatment of cancer andchronic infection.

WO 2016/028656 discloses anti-TIGIT antibodies, as well as use of theseantibodies in the treatment of diseases such as cancer and infectiousdisease.

There is an unmet need to provide additional and more effective,specific, safe and/or stable agents that alone or in combination withother agents, allow cells of the immune system to attack tumor cells byinhibiting the suppresser activity of human TIGIT.

SUMMARY OF THE INVENTION

The present invention provides agents that recognize the immune cellinhibitory receptor “T-Cell immunoglobulin and ITIM domain” (TIGIT) andinhibit its suppressive activity on lymphocytes such as natural killer(NK) cells and T-cells. These agents are for example antibodies andfragment thereof, characterized by having unique sets of CDR sequences,exceptional high affinity and high specificity to human TIGIT and areuseful in cancer immunotherapy for combating tumor immune evasion, asstand-alone therapy and in combination with other anti TIGIT antibodiesand/or other anti-cancer agents.

Some of the monoclonal antibodies (mAbs) of the present inventionexhibits absolute specificity to human TIGIT, as compared to mouse TIGITfor example, and affinity to the human TIGIT protein, which is higherthan the affinity of the natural ligand, PVR (CD155) to human TIGIT.This makes these superior mAbs valuable candidates for use in cancerimmune-therapy, enabling administration of lower doses with fewer sideeffects.

Some of the monoclonal antibodies of the present invention are capablenot only to interfere with the binding of human TIGIT to its main ligandPVR (CD155) but also to inhibit, at least to some extent, the binding ofTIGIT to at least one of its other ligands, such as CD112 and CD113.

Some of the anti-TIGIT monoclonal antibodies described herein havesynergistic effect when combined with additional anti-cancer agents,such as other immunomodulatory proteins or receptor inhibitors.Non-limiting examples are mAbs specific to cytotoxicT-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), andepidermal growth factor receptor inhibitors (EGFR).

The present invention provides, according to one aspect, an isolatedantibody, or a fragment thereof comprising at least the antigen bindingportion that recognizes human TIGIT with an affinity of at least 10⁻⁸M,and inhibits its interaction with at least one ligand.

According to some embodiments, the isolated antibody is a monoclonalantibody (mAb) or a fragment thereof.

According to some embodiments, the isolated monoclonal antibody orfragment comprises the complementarity determining region (CDR)sequences of a monoclonal antibody denoted VSIG9#1 (or Vsig9.01),namely, the three CDR sequences contained in heavy chain variable regionset forth in SEQ ID NO:7 and the three CDR sequences contained in lightchain variable region set forth in SEQ ID NO:8. Determination of CDRsequences can be made according to any method known in the art,including but not limited to the methods known as KABAT, Chothia andIMGT. A selected set of CDRs may include sequences identified by morethan one method, namely, some CDR sequences may be determined usingKABAT and some using IMGT, for example.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises heavy chain CDR1 (^(HC)CDR1) sequence comprising asequence selected from the group consisting of: GYTFTSYGIS (SEQ IDNO:1), and TSYGIS (SEQ ID NO:11), heavy chain CDR2 (^(HC)CDR2)comprising the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chainCDR3 (^(HC)CDR3) comprising the sequence: KGPYYTKNEDY (SEQ ID NO:3),light chain CDR1 (^(LC)CDR1) comprising the sequence: RASEHIYYSLA (SEQID NO:4), light chain CDR2 (^(LC)CDR2) comprising the sequence: NANSLED(SEQ ID NO:5), and light chain CDR3 (^(LC)CDR3) comprising the sequence:KQAYDVPRT (SEQ ID NO:6), and analogs thereof comprising no more than 5%amino acid substitution, deletion and/or insertion in the hypervariableregion (HVR) sequence.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises heavy chain CDR1 (^(HC)CDR1) sequence having thesequence GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2 (^(HC)CDR2) havingthe sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3(^(HC)CDR3) having the sequence: KGPYYTKNEDY (SEQ ID NO:3), light chainCDR1 (^(LC)CDR1) having the sequence: RASEHIYYSLA (SEQ ID NO:4), lightchain CDR2 (^(LC)CDR2) having the sequence: NANSLED (SEQ ID NO:5), andlight chain CDR3 (^(LC)CDR3) having the sequence: KQAYDVPRT (SEQ IDNO:6), and analogs thereof comprising no more than 5% amino acidsubstitution, deletion and/or insertion in the hypervariable region(HVR) sequence.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises heavy chain variable region having thesequence: QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNEDYWG QGTILTVSS (SEQID NO:7), or an analog or derivative thereof having at least 90%sequence identity with the heavy chain variable region sequence.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises light chain variable region having thesequence: DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRT FGGGTKLEIKRADAAPTVS (SEQ IDNO:8), or an analog thereof having at least 90% sequence identity withthe light chain variable region sequence.

According to a specific embodiment, the isolated monoclonal antibody orfragment thereof comprises a heavy chain variable region having thesequence: QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNEDYWG QGTILTVSS (SEQID NO:7), and a light chain variable region having the sequence:DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRTFGGGTKLEIKRADAA PTVS (SEQ IDNO:8), or an analog thereof having at least 90% sequence identity withthe light and/or heavy chain sequence.

The invention also encompasses antibody or antibody fragment capable ofbinding with high affinity to an epitope within the human TIGIT proteinto which mAb VSIG9#1 binds.

According to other embodiments, the isolated monoclonal antibodycomprises the complementarity determining region (CDR) sequences of amonoclonal antibody denoted #4 (or 258-cs1#4), namely, the three CDRsequences contained in heavy chain variable region set forth in SEQ IDNO:18 and the three CDR sequences contained in light chain variableregion set forth in SEQ ID NO:19.

According to some specific embodiments the isolated monoclonal antibodycomprises heavy chain CDR1 (^(HC)CDR1) comprising the sequence IYCIH(SEQ ID NO:12), heavy chain CDR2 (^(HC)CDR2) comprising the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 (^(HC)CDR3)comprising the sequence: SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1(^(LC)CDR1) comprising the sequence: RASQEISGYLN (SEQ ID NO:15), lightchain CDR2 (^(LC)CDR2) comprising the sequence: AASTLDS (SEQ ID NO:16),and light chain CDR3 (^(LC)CDR3) comprising the sequence: LQYASYPRT (SEQID NO:17), and analogs thereof comprising no more than 5% amino acidsubstitution, deletion and/or insertion in the hypervariable region(HVR) sequence.

According to some specific embodiments the isolated monoclonal antibodycomprises heavy chain CDR1 (^(HC)CDR1) having the sequence IYCIH (SEQ IDNO:12), heavy chain CDR2 (^(HC)CDR2) having the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 (^(HC)CDR3) havingthe sequence: SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 (^(LC)CDR1)having the sequence: RASQEISGYLN (SEQ ID NO:15), light chain CDR2(^(LC)CDR2) having the sequence: AASTLDS (SEQ ID NO:16), and light chainCDR3 (^(LC)CDR3) having the sequence: LQYASYPRT (SEQ ID NO:17), andanalogs and derivative thereof.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises heavy chain variable region having thesequence: QVQLLQPGAELVKPGASVKLSCKASGYTFTIYCIHWVKQRPGQGLEWIGEISPSNGRTIYNEKFKNKATLTIDKSSTTAYMQLSSLTSEDSAVYCCAISDGYDGYYFDYWGQ GTTLTVSS (SEQID NO:18), or an analog or derivative thereof having at least 90%sequence identity with the heavy chain sequence.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises light chain variable region having thesequence: DIQMTQSPSSLSASLGERVSLTCRASQEISGYLNWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISRLESEDFADYYCLQYASYPRTFGGGTKLEIK (SEQ ID NO:19), or ananalog or derivative thereof having at least 90% sequence identity withthe light chain sequence.

The invention also encompasses antibody or antibody fragment capable ofbinding with high affinity to an epitope within the human TIGIT proteinto which mAb #4 binds.

Analogs and derivatives of the isolated mAb antibodies, and the antibodyfragments described above, are also within the scope of the invention.In particular analogs or isolated mAbs or fragment thereof comprising atleast one variable region set forth in a sequence selected from thegroup consisting of: SEQ ID No: 7, 8, 18 and 19 are also within thescope of the present invention.

According to some embodiments, the antibody or antibody fragment analoghave at least 95% sequence identity with the hypervariable region of thereference antibody sequence, or at least 90% sequence identity with theheavy or light chain variable regions of the reference antibody.

According to certain embodiments, the analog or derivative of theisolated antibody or fragment thereof has at least 91, 92, 93, 94, 95,96, 97, 98 or 99% sequence identity with a variable region of thereference antibody sequence. Each possibility represents a separateembodiment of the invention.

According to another aspect the present invention provides a monoclonalantibody or an antibody fragment that recognizes TIGIT, comprising anantigen binding domain (ABD) comprising three CDRs of a light chain andthree CDRs of a heavy chain, wherein said CDRs have at least 90%sequence identity or similarity with the CDRs of the ABD of: (i) amonoclonal mouse antibody (herein identified as VSIG9#1 or Vsig9.01clone) comprising a heavy chain variable region of SEQ ID NO:7 and alight chain variable region of SEQ ID NO:8; or (ii) a monoclonal mouseantibody (herein identified #4 or 258-CS1#4 clone) comprising a heavychain variable region of SEQ ID NO:18 and a light chain variable regionof SEQ ID NO:19. According to some embodiments, the CDRs have at least91%, at least 92%, at least 93% or at least 94% sequence identity orsimilarity with those of VSIG9#1 or 258-CS1#4. According to otherembodiments, the ABD has at least 95%, at least 96%, or at least 97%, atleast 98% or at least 99% sequence identity or similarity with VSIG9#1or 258-CS1#4.

According to some embodiments, the antibody or antibody fragmentaccording to the invention comprises a heavy chain variable region setforth in SEQ ID NO:7, or an analog having at least 95% sequencesimilarity with said sequence. According to other embodiments, theantibody or antibody fragment according to the invention comprises aheavy chain variable region set forth in SEQ ID NO:18, or an analoghaving at least 95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragmentcomprises a light chain variable region set forth in SEQ ID NO:8, or ananalog having at least 95% sequence similarity with said sequence.According to other embodiments, the antibody or antibody fragmentcomprises a light chain variable region set forth in SEQ ID NO:19, or ananalog having at least 95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragmentcomprises a heavy chain and a light chain, wherein: (i) the heavy chaincomprises SEQ ID NO:7 and the light chain comprises SEQ ID NO8; or (ii)the heavy chain comprises SEQ ID NO:18 and the light chain comprises SEQID NO:19. Analogs of the antibodies or fragments, having at least 95%sequence similarity with said heavy or light chains are also included.

According to some embodiments, the analog has at least 96, 97, 98 or 99%sequence identity with an antibody light or heavy chain variable regionsdescribed above. According to some embodiments, the analog comprises nomore than one amino acid substitution, deletion or addition to one ormore CDR sequences of the hypervariable region, namely, any one of theCDR sequences set forth in SEQ ID Nos: 1-6 and 12-17. According to someembodiments, the amino acid substitution is a conservative substitution.Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the antibody or antibody fragmentcomprises a hypervariable region (HVR) having light and heavy chainregions defined above, in which 1, 2, 3, 4, or 5 amino acids weresubstituted, deleted and/or added. Each possibility represents aseparate embodiment of the invention. According to specific embodiments,the antibody or antibody fragment comprises a hypervariable regionhaving a set of CDR sequences selected from SEQ ID NOs.: 1-6 and 12-17,in which no more than one amino acid is substituted, deleted or added toat least one CDR sequence. According to some embodiments, the amino acidsubstitution is a conservative substitution. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragment iscapable of recognizing TIGIT protein expressed on T-cells.

According to some embodiments, the antibody or antibody fragment iscapable of recognizing human TIGIT protein expressed on dendritic or NKcells.

According to some embodiments, the antibody or antibody fragment iscapable of recognizing human TIGIT protein expressed on T-regulatorycells (Treg).

According to some embodiments, the antibody or antibody fragment iscapable of recognizing human TIGIT protein expressed on CD8+ tumorinfiltrating lymphocytes (TILs).

According to some embodiments, the antibody or antibody fragment iscapable of inhibiting human TIGIT binding to a ligand expressed onT-cells.

According to some embodiments, the antibody or antibody fragment iscapable of inhibiting human TIGIT binding to ligand expressed ondendritic or NK cells.

According to yet other embodiments, the antibody or antibody fragment iscapable of inhibiting human TIGIT binding to ligand expressed on tumorcells.

According to some embodiments, the antibody or antibody fragment iscapable of inhibiting binding of human TIGIT to a ligand selected fromthe group consisting of: PVR (CD155), PVRL2 (CD112/Nectin2), PVRL3(CD113) and any combination thereof.

According to some embodiments, the isolated antibody or fragment thereofbinds to human TIGIT protein with a dissociation constant (Kd) of atleast about 50 nM. According to some embodiments the isolated antibodyor fragment thereof binds to human TIGIT protein with a binding affinityof at least 1 nM. Thus, according to some embodiments, antibody orantibody according to the inventor has an affinity to human TIGIT of atleast about 5×10⁻⁸M. According to other embodiments, an antibody orantibody fragment binds with an affinity of 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M or even higher to human TIGIT. Eachpossibility represents a separate embodiment of the invention.

According to a specific embodiment, the mAb is selected from the groupconsisting of: non-human antibody, humanized antibody, human antibody,chimeric antibody, bispecific antibody and an antibody fragmentcomprising at least the antigen-binding portion of an antibody.According to a specific embodiment, the antibody fragment is selectedfrom the group consisting of: Fab, Fab′, F(ab′)₂, Fd, Fd′, Fv, dAb,isolated CDR region, single chain antibody (scab), “diabodies”, and“linear antibodies”. Each possibility represents a separate embodimentof the present invention.

According to some embodiments, the antibody is a bispecific antibody orbispecific antibody fragment, capable of binding to two differentepitopes or antigens.

According to some embodiments, a bispecific mAb or bispecific mAbfragment comprises two different hyper variable regions (HVR), eachcomprising a different set of CDR sequences.

According to some embodiments the bispecific mAb or fragment comprisesthe binding domains of two different anti-TIGIT antibodies. Each HVR ofa bispecific mAb or fragment according to these embodiments is cable ofbinding to a different epitope of the human TIGIT protein.

According to some embodiments, one HVR of a bispecific mAb or fragmentcomprises the CDRs contained in heavy chain sequence set forth in SEQ IDNO:7 and light chain sequence set for the in SEQ ID NO:8 of themonoclonal antibody VSIG9#1; and the second HVR comprises the CDRscontained in heavy chain sequence set for the in SEQ ID NO:18 and lightchain sequence set forth in SEQ ID NO:19 of the monoclonal antibody #4(or 258-cs1#4).

According to some embodiments, one HVR of a bispecific mAb or fragmentthereof comprises the CDRs: SEQ ID NO:1 or SEQ ID NO:11, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and the secondHVR comprises the CDRs: SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16 and SEQ ID NO:17.

According to other embodiments, one HVR of a bispecific antibody orbispecific antibody fragment according to the invention binds to humanTIGIT and the second HVR binds to another protein, such as a proteininvolved in immune regulation or a tumor antigen. According to somespecific embodiments, the second HVR of the bispecific antibody binds acheckpoint molecule.

According to some embodiments, the antibody or antibody fragmentcomprises a framework sequence selected from the group consisting of:mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, humanIgG3, and human IgG4. Each possibility represents a separate embodimentof the present invention.

According to other embodiments, the antibody is a humanized antibody orthe antibody fragment is a fragment of a humanized antibody.

According to some embodiments, the humanized antibody or antibodyfragment comprises a framework sequence selected from the groupconsisting of: human IgG1, human IgG2, human IgG3, and human IgG4. Eachpossibility represents a separate embodiment of the present invention.

According to yet other embodiments, an antibody conjugate comprising atleast one antibody or antibody fragment that recognizes TIGIT andinhibits binding to its ligand is provided wherein said antibody orantibody fragment comprises the complementarity determining regions(CDRs) sequences: (i) heavy chain CDR1 having a sequence selected from:GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO:11), heavy chain CDR2having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3having the sequence: KGPYYTKNEDY (SEQ ID NO:3), light chain CDR1 havingthe sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having thesequence: NANSLED (SEQ ID NO:5), and light chain CDR3 having thesequence: KQAYDVPRT (SEQ ID NO:6); or (ii) heavy chain CDR1 having thesequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having the sequence:SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having the sequence:RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having the sequence:AASTLDS (SEQ ID NO:16), and light chain CDR3 having the sequence:LQYASYPRT (SEQ ID NO:17).

According to some embodiments, the conjugate comprises a carrierprotein.

Polynucleotide sequences encoding monoclonal antibodies, having highaffinity and specificity for TIGIT, as well as vectors and host cellscarrying these polynucleotide sequences, are provided according toanother aspect of the present invention.

According to some embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain capable of binding to an epitopewithin the human TIGIT protein to which binds: (i) a mouse monoclonalantibody (herein identified as VSIG9#1) having a heavy chain variableregion of SEQ ID NO:7 and a light chain variable region of SEQ ID NO:8;or (ii) a mouse monoclonal antibody (herein identified as 258-CS1#4)having a heavy chain variable region of SEQ ID NO:18 and a light chainvariable region of SEQ ID NO:19.

According to some embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain comprising the sequence set forthin SEQ ID NO:7. According to some embodiments, the polynucleotidesequence encodes an antibody or antibody fragment or chain comprisingthe sequence set forth in SEQ ID NO:8.

According to other embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain comprising the sequence set forthin SEQ ID NO:18. According to additional embodiments, the polynucleotidesequence encodes an antibody or antibody fragment or chain comprisingthe sequence set forth in SEQ ID NO:19.

According to yet some embodiments, a polynucleotide sequence accordingto the invention encodes an antibody or antibody fragment or chaincomprising the six CDR sequences: (i) heavy chain CDR1 sequence selectedfrom: GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO:11), heavy chainCDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chainCDR3 having the sequence: KGPYYTKNEDY (SEQ ID NO:3), light chain CDR1having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 havingthe sequence: NANSLED (SEQ ID NO:5), and light chain CDR3 having thesequence: KQAYDVPRT (SEQ ID NO:6); or (ii) heavy chain CDR1 having thesequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having the sequence:SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having the sequence:RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having the sequence:AASTLDS (SEQ ID NO:16), and light chain CDR3 having the sequence:LQYASYPRT (SEQ ID NO:17).

According to some embodiments, a polynucleotide sequences defined aboveencodes a molecule selected from the group consisting of: an antibody anantibody fragment comprising at least an antigen-binding portion, anantibody conjugate comprising said antibody or antibody fragment, and abispecific antibody. Each possibility represents a separate embodimentof the present invention.

According to some embodiments, a polynucleotide sequence is providedcomprising the sequence of a monoclonal antibody heavy chain variableregion set forth in SEQ ID NO:9, or a variant thereof having at least90% sequence identity:

(SEQ ID NO: 9) CAGGTGCAGCTGCAGGAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTATCCCAGAAGTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCGTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAAAGGGACCCTACTATACTAAGAACGAGGACTACTGGGGCCAAGGCACCATTCTCAC AGTCTCCTCA.

According to some embodiments, a polynucleotide sequence is providedcomprising the sequence of a monoclonal antibody heavy chain variableregion set forth in SEQ ID NO:20, or a variant thereof having at least90% sequence identity:

(SEQ ID NO: 20) CAGGTCCAACTGCTGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCATCTACTGTATACACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAGTCCTAGCAACGGTCGTACTATCTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTATAGACAAATCCTCCACCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTGCTGTGCAATATCGGATGGTTACGACGGATACTACTTTGACTACTGGGGCCAAGGCACCACTCTCAC AGTCTCCTCA.

According to some embodiments, a polynucleotide sequence is providedcomprising the sequence of a monoclonal antibody light chain variableregion set forth in SEQ ID NO:10, or a variant thereof having at least90% sequence identity:

(SEQ ID NO: 10) GACATCCAGATGACTCAGTCTCCAGCCTCCCTGGCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGCACATTTACTACAGTTTAGCATGGTATCAGCAGAAGCAAGGGAAATCTCCTCAGCTCCTGATCTATAATGCAAACAGCTTGGAAGATGGTGTCCCATCGAGGTTCAGTGGCAGTGGATCTGGGACACAATATTCTATGAAGATCAACAGCATGCAGCCTGAAGATACCGCAACTTATTTCTGTAAACAGGCTTATGACGTTCCTCGGACCTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGG CTGATGCTGCACCAACTGTATCC.

According to some embodiments, a polynucleotide sequence is providedcomprising the sequence of a monoclonal antibody light chain variableregion set forth in SEQ ID NO:21, or a variant thereof having at least90% sequence identity:

(SEQ ID NO: 21) GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAACTGGCTTCAGCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTAGATTCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGACTTGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAATATGCTAGTTATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

The present invention provides, according to some embodiments, apolypeptide comprising at least one sequence encoded by at least onepolynucleotide sequence disclosed above.

In a further aspect the present invention provides a nucleic acidconstruct comprising a nucleic acid molecule encoding at least oneantibody chain or fragment thereof according to the present invention.According to some embodiments the nucleic acid construct is a plasmid.

According to some embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO:9 or SEQ ID NO:20.

According to other embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO:10 or SEQ ID NO:21.

In still another aspect the present invention provides a hybridoma cellcapable of producing an antibody or an antibody fragment comprising thespecific CDR sequences and/or specific heavy and light chain variableregions defined above.

According to some embodiments, a hybridoma cell is provided comprisingat least one polynucleotide sequence disclosed above.

According to some embodiments, the hybridoma is a cable of producing amonoclonal antibody comprising the six complementarity determiningregions (CDRs) sequences: (i) heavy chain CDR1 sequence selected fromGYTFTSYGIS (SEQ ID NO:1) and TSYGIS (SEQ ID NO:11), heavy chain CDR2having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3having the sequence: KGPYYTKNEDY (SEQ ID NO:3), light chain CDR1 havingthe sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having thesequence: NANSLED (SEQ ID NO:5), and light chain CDR3 having thesequence: KQAYDVPRT (SEQ ID NO:6); or (ii) heavy chain CDR1 having thesequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having the sequence:SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having the sequence:RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having the sequence:AASTLDS (SEQ ID NO:16), and light chain CDR3 having the sequence:LQYASYPRT (SEQ ID NO:17).

Antibodies or fragments thereof according to the present invention maybe attached to a cytotoxic moiety, a radioactive moiety, or anidentifiable moiety.

The present invention provides, according to another aspect, apharmaceutical composition comprising as an active ingredient, at leastone antibody, antibody fragment or conjugates thereof, that recognizesTIGIT with high affinity and specificity and inhibits its interactionwith one of its ligands, and optionally at least one pharmaceuticalacceptable excipient, diluent, salt or carrier.

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or a fragment thereof which is capable of bindingto an epitope within the human TIGIT protein to which binds a mousemonoclonal antibody selected from the group consisting of: (i) anantibody (herein identified as VSIG9#1 or Vsig9.01) having a heavy chainvariable region of SEQ ID NO:7 and a light chain variable region of SEQID NO:8; and (ii) an antibody (herein identified as #4 or 258-CS1#4)having a heavy chain variable region of SEQ ID NO:18 and a light chainvariable region of SEQ ID NO:19.

According to some embodiments, the monoclonal antibody or antibodyfragment thereof comprises the six CDRs: (i) heavy chain CDR1 sequenceselected from: GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO:11),heavy chain CDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2),heavy chain CDR3 having the sequence: KGPYYTKNEDY(SEQ ID NO:3), lightchain CDR1 having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chainCDR2 having the sequence: NANSLED (SEQ ID NO:5), and light chain CDR3having the sequence: KQAYDVPRT (SEQ ID NO:6); or (ii) heavy chain CDR1having the sequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having thesequence: EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having thesequence: SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having thesequence: RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having thesequence: AASTLDS (SEQ ID NO:16), and light chain CDR3 having thesequence: LQYASYPRT (SEQ ID NO:17).

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or fragment thereof comprising a heavy chainvariable region having the sequence:

(i) (SEQ ID NO: 7) QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKG PYYTKNEDYWGQGTILTVSS;or  (ii) (SEQ ID NO: 18)QVQLLQPGAELVKPGASVKLSCKASGYTFTIYCIHWVKQRPGQGLEWIGEISPSNGRTIYNEKFKNKATLTIDKSSTTAYMQLSSLTSEDSAVYCCAISD GYDGYYFDYWGQGTTLTVSS.

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or fragment thereof comprising a light chainvariable region having the sequence:

(i) (SEQ ID NO: 8) DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRT FGGGTKLEIKRADAAPTVS; or (ii) (SEQ ID NO: 19)DIQMTQSPSSLSASLGERVSLTCRASQEISGYLNWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISRLESEDFADYYCLQYASYPRTF GGGTKLEIK.

According to a specific embodiment, the pharmaceutical compositioncomprises a monoclonal antibody or fragment thereof comprising a heavychain variable region having the sequence:

(SEQ ID NO: 7) QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNEDYWGQGTILTVSS,

and a light chain variable region having the sequence:

(SEQ ID NO: 8) DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRT FGGGTKLEIKRADAAPTVS.

According to an additional specific embodiment, the pharmaceuticalcomposition comprises a monoclonal antibody or fragment thereofcomprising a heavy chain variable region having the sequence:

(SEQ ID NO: 18) QVQLLQPGAELVKPGASVKLSCKASGYTFTIYCIHWVKQRPGQGLEWIGISPSNGRTIYNEKFKNKATLTIDKSSTTAYMQLSSLTSED SAVYCCAISDGYDGYYFDYWGQGTTLTVSS

and a light chain variable region having the sequence:

(SEQ ID NO: 19) DIQMTQSPSSLSASLGERVSLTCRASQEISGYLNWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISRLESEDFADYYCLQYASYPRTF GGGTKLEIK.

According to some embodiments, the pharmaceutical composition comprisesat least one bispecific antibody comprising two different HVR regions,wherein at least one of the HVR binds with high affinity and selectivityto human TIGIT.

According to some embodiments, the second HVR binds to another epitopeon human TIGIT or to a different antigen.

According to some embodiments, one HVR comprises the CDR sequences ofthe antibody denoted VSIG9#1 (or Vsig9.01) and the second HVR comprisesthe CDR sequences of the antibody denoted 258-cs1#4 (also denoted #4).

According to some embodiments, one HVR comprises the six CDR sequencesof the antibody denoted VSIG9#1 or the six CDRs the antibody denoted258-cs1#4, and the second HVR comprises six different CDRs capable ofbinding human TIGIT or a different antigen.

According to some embodiments, the pharmaceutical composition comprisesa combination of at least two antibodies, or antibody fragments, whichrecognizes human TIGIT, wherein at least one of the antibodies has highaffinity and selectivity to human TIGIT. According to some embodiments,the at least one antibody or antibody fragment that has high affinityand selectivity to human TIGIT comprises the CDR sequences of theantibody denoted VSIG9#1 (or Vsig9.01) or 258-cs1#4 (also denoted #4).

According to some specific embodiments, one monoclonal antibody orantibody fragment comprises the CDR sequences of the antibody denotedVSIG9#1 and the second antibody or antibody fragment comprises the CDRsequences of the antibody denoted 258-cs1#4 (or #4).

According to yet other embodiments, the composition comprises one mAb orfragment that specifically binds TIGIT, according to the invention, andone mAb or fragment that specifically binds a different antigen, suchas, cell-receptor, tumor antigen or immunomodulatory.

Also provided are pharmaceutical compositions, comprising at least oneantibody, antibody fragment or antibody conjugate according to theinvention, for use in restoring NK cytotoxicity by inhibiting binding ofTIGIT ligand to NK cells.

According to some embodiments, the pharmaceutical composition accordingto the present invention is for use in cancer immunotherapy or inenhancing immune response.

According to yet another aspect, the present invention provides a methodof inhibiting binding of human TIGIT to at least one ligand by using amonoclonal antibody or antibody fragment defined above.

According to some embodiments, the antibody or antibody fragment iscapable of inhibiting binding of TIGIT to a ligand selected from thegroup consisting of: PVR (CD155), PVRL2 (CD112), PVRL3 (CD113), and anycombination thereof.

According to some embodiments, a method of restoring NK cytotoxicity isprovided by inhibiting binding of TIGIT to at least one ligand expressedon NK cells, comprising administering to a subject in need thereof apharmaceutical composition comprising at least one antibody, antibodyfragment or antibody conjugate that recognizes human TIGIT with highaffinity and specificity.

According to some embodiments, the antibody, antibody fragment orantibody conjugate is capable of inhibiting human TIGIT binding to aligand expressed on T-cells.

According to some embodiments, the antibody, antibody fragment orantibody conjugate is capable of inhibiting human TIGIT binding to aligand expressed on dendritic or NK cells.

According to yet other embodiments, the antibody, antibody fragment orantibody conjugate is capable of inhibiting human TIGIT binding toligand expressed on tumor cells.

The invention provides in another aspect methods for enhancing immuneresponse in a subject in need thereof comprising administering to saidsubject a therapeutically effective amount of a monoclonal antibody,antibody fragment or antibody conjugate defined above.

According to yet another aspect, the present invention provides a methodof treating cancer comprising administering to a subject in needthereof, a pharmaceutical composition comprising at least one antibody,antibody fragment or conjugate thereof, that recognizes human TIGIT withhigh affinity and specificity and capable of inhibiting its binding toits ligand.

According to some embodiments, the cancer is selected from the groupconsisting of: lung, thyroid, breast, colon, melanoma, prostate,hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma,myeloid, ovarian, uterus, sarcoma, biliary, non-small-cell lung andendometrial cells cancer. Each possibility represents a separateembodiment of the invention.

According to some embodiments, the cancer is selected from the groupconsisting of: melanoma, breast cancer, non-small-cell lung and colonand hepatic (liver) cancer. Each possibility represents a separateembodiment of the invention.

According to some specific embodiments, the cancer is melanoma.

According to some embodiments, the cancer is a solid cancer. Accordingto some specific embodiments, the solid cancer is selected from thegroup consisting of melanoma (skin), lung, colon, breast, uterine, andrenal cancer.

According to some embodiments, the cancer is leukemia. According to somespecific embodiments, the cancer is acute myeloid leukemia (AML).

According to some embodiments, the antibody in the pharmaceuticalcomposition administered is selected from the group consisting of: (i) amonoclonal antibody comprising the CDR sequences contained in heavychain variable region set forth in SEQ ID NO:7 and the CDR sequencescontained in light chain variable region set forth in SEQ ID NO:8; or(ii) a monoclonal antibody comprising the CDR sequences contained inheavy chain variable region set forth in SEQ ID NO:18 and the CDRsequences contained in light chain variable region set forth in SEQ IDNO:19.

According to some specific embodiments, the monoclonal antibody in thepharmaceutical composition administered comprises: heavy chain CDR1having the sequence: GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2 havingthe sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3 havingthe sequence: KGPYYTKNEDY (SEQ ID NO:3), light chain CDR1 having thesequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having thesequence: NANSLED (SEQ ID NO:5), and light chain CDR3 having thesequence: KQAYDVPRT (SEQ ID NO:6).

According to other specific embodiments, the monoclonal antibody in thepharmaceutical composition administered comprises: heavy chain CDR1having the sequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having thesequence: EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having thesequence: SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having thesequence: RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having thesequence: AASTLDS (SEQ ID NO:16), and light chain CDR3 having thesequence: LQYASYPRT (SEQ ID NO:17).

According to some embodiments, the method of treating cancer comprisesadministering or performing at least one additional anti cancer therapy.According to certain embodiments, the additional anticancer therapy issurgery, chemotherapy, radiotherapy, or immunotherapy.

According to some embodiments, the method of treating cancer comprisesadministration of a monoclonal antibody that recognizes human TIGIT withhigh affinity and specificity and an additional anti-cancer agent.According to some embodiments, the additional anti-cancer agent isselected from the group consisting of: immune-modulator, activatedlymphocyte cell, kinase inhibitor and chemotherapeutic agent.

According to some embodiments, the additional immune-modulator is anantibody, antibody fragment or antibody conjugate that binds to adifferent epitope on human TIGIT to which the monoclonal antibodydenoted VSIG9#1 (or Vsig9.01) binds.

According to other embodiments, the additional immune-modulator is anantibody, antibody fragment or antibody conjugate that binds to anantigen other than human TIGIT.

According to some embodiments, the additional immune-modulator is anantibody against an immune checkpoint molecule. According to someembodiments, the additional immune modulator is an antibody against animmune checkpoint molecule selected from the group consisting of humanprogrammed cell death protein 1 (PD-1), PD-L1 and PD-L2,carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1),lymphocyte activation gene 3 (LAG3), CD137, OX40 (also referred to asCD134), killer cell immunoglobulin-like receptors (KIR), and anycombination thereof. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the method of treating cancer comprisesadministration of a pharmaceutical composition comprising a monoclonalantibody that recognizes human TIGIT according to the invention and ananti-PD-1 antibody.

According to some embodiments, the method of treating cancer comprisesadministration of a pharmaceutical composition comprising a monoclonalantibody that recognizes human TIGIT according to the invention and ananti-CTLA-4 antibody.

According to some embodiments, the method of treating cancer comprisesadministering the pharmaceutical composition as part of a treatmentregimen comprising administration of at least one additional anti-canceragent.

According to some embodiments, the anti-cancer agent is selected fromthe group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil,mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine,vinblastine, vinorelbine, carmustine, lomustine, chlorambucil,cyclophosphamide, cisplatin, carboplatin, ifosamide, mechlorethamine,melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin,doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide,teniposide and combinations thereof. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, the anti-cancer agent is epidermal growthfactor receptor (EGFR) inhibitor. According to some embodiments, theEGFR inhibitor is selected from the group consisting of: Cetuximab(Erbitux®), Panitumumab (Vectibix®), and necitumumab (Portrazza®).According to some embodiments, the EGFR inhibitor is Cetuximab(Erbitux®).

The present invention thus provides a method of increasing orstimulating an immune response by administering a pharmaceuticalcomposition comprising at least one antibody, conjugate, or fragmentthereof that recognizes TIGIT and inhibits its binding to its ligand.

The present invention further comprises, according to another aspect, amethod of determining or quantifying the expression of TIGIT, the methodcomprising contacting a biological sample with an antibody or antibodyfragment, and measuring the level of complex formation, wherein theantibody or antibody fragment comprises the complementarity determiningregions (CDRs) selected from the group consisting of: (i) heavy chainCDR1 having the sequence: GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3having the sequence: KGPYYTKNEDYV (SEQ ID NO:3), light chain CDR1 havingthe sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having thesequence: NANSLED (SEQ ID NO:5), and light chain CDR3 having thesequence: KQAYDVPRT (SEQ ID NO:6); and (ii) heavy chain CDR1 having thesequence IYCIH (SEQ ID NO:12), heavy chain CDR2 having the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13), heavy chain CDR3 having the sequence:SDGYDGYYFDY (SEQ ID NO:14), light chain CDR1 having the sequence:RASQEISGYLN (SEQ ID NO:15), light chain CDR2 having the sequence:AASTLDS (SEQ ID NO:16), and light chain CDR3 having the sequence:LQYASYPRT (SEQ ID NO:17).

Determining and quantifying methods may be performed in-vitro or ex-vivoaccording to some embodiments or may be used in diagnosing conditionsassociated with expression of TIGIT. The antibodies according to thepresent invention may be also used to configure screening methods. Forexample, an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed for measuring levels of secreted orcell-associated polypeptide using the antibodies and methods known inthe art.

According to one embodiment a method is provided for detecting orquantifying the presence of TIGIT, comprising the steps of:

-   -   i. incubating a sample with an antibody specific to TIGIT or an        antibody fragment thereof comprising at least an antigen-binding        portion;    -   ii. detecting the bound TIGIT using a detectable probe.

According to some embodiments, the method further comprises the stepsof:

-   -   iii. comparing the amount of (ii) to a standard curve obtained        from a reference sample containing a known amount of TIGIT; and    -   iv. calculating the amount of the TIGIT in the sample from the        standard curve.

According to some particular embodiments the sample is a body fluid.

According to some embodiments, the method is performed in-vitro orex-vivo.

A kit for measuring the expression of TIGIT in biological sample is alsoprovided comprising at least one antibody or antibody fragmentcomprising the complementarity determining regions (CDRs) selected fromthe group consisting of: (i) heavy chain CDR1 having the sequence:GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2 having the sequence:EIYPRSGNTYYNEKFKG (SEQ ID NO:2), heavy chain CDR3 having the sequence:KGPYYTKNEDY (SEQ ID NO:3), light chain CDR1 having the sequence:RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having the sequence: NANSLED(SEQ ID NO:5), and light chain CDR3 having the sequence: KQAYDVPRT (SEQID NO:6); and (ii) heavy chain CDR1 having the sequence IYCIH (SEQ IDNO:12), heavy chain CDR2 having the sequence: EISPSNGRTIYNEKFKN (SEQ IDNO:13), heavy chain CDR3 having the sequence: SDGYDGYYFDY (SEQ IDNO:14), light chain CDR1 having the sequence: RASQEISGYLN (SEQ IDNO:15), light chain CDR2 having the sequence: AASTLDS (SEQ ID NO:16),and light chain CDR3 having the sequence: LQYASYPRT (SEQ ID NO:17).

In some embodiments, the invention provides a method of diagnosing,assessing the severity or staging an immune-related disease or aproliferative disease comprising determining the expression or activityof TIGIT in a sample from a subject using an antibody according to thepresent invention or a fragment or conjugate thereof, and comparing theexpression or activity of TIGIT to a reference amount of TIGITexpression or activity. Said reference amount may be obtained from asample taken from a normal subject, from the same subject while being ina different stage of the disease or is determined from clinical data ofa large population of subjects.

Antibodies, antibody fragments or conjugates thereof, according to thepresent invention may be used in any diagnostic, therapeutic orprophylactic method that utilizes binding to the human protein TIGIT, aslong as they are capable of specifically binding to said protein andinhibiting it's binding to at least one ligand.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Expression of TIGIT on effector immune cells.

FIG. 1A. FACS histogram plot of 2*10⁵ YTS cells (Left) or YTS cellsover-expressing TIGIT (right). Left gray filled curves represent thestaining with control mouse IgG (mIgG). Right empty curves represent thestaining with two anti human TIGIT mAbs as indicated (VSIG9#1 and258-CS1#4).

FIG. 1B. FACS histogram plots of TIGIT expression on NK cells of twohealthy donors (I and II as indicated). Left gray filled curvesrepresent the staining with control mIgG. Right empty curve representsthe staining with anti TIGIT (Clone VSIG9#1).

FIG. 1C. FACS histogram plots of TIGIT expression on CD8+ TILs cellsfrom two melanoma patients (TIL-I and TIL-II). Left gray filled curverepresents the staining with control mIgG. Right empty curve representsthe staining with anti TIGIT (Clone VSIG9#1).

FIGS. 2A-2B. Inhibition of TIGIT binding by mAb VSIG9#1:

FIG. 2A. FACS histogram plot of HepG2 cells (that express high levels ofPVR, Nectin-2 and Nectin-3) that were incubated with hTIGIT-Fc (1),hTIGIT-Fc with control mIgG (2) or hTIGIT-Fc with anti-TIGIT mAb VSIG9#1at the indicated concentrations (3 and 4).

FIG. 2B. FACS histogram plot of TIGIT ligands (empty histograms)expressed by HepG2 cells indicated at the individual plots. Grey filledhistograms represent staining with control mIgG only.

FIGS. 3A-3D. Blocking of TIGIT leads to enhanced killing activity.

FIG. 3A. Specific killing activity was measured for ³⁵S labeled721.221-PVR cells that were incubated with YTS-TIGIT cells (at ratio of1:10) and with either control mIgG, (left bar) or anti TIGIT-VSIG9#1(right bar). *p<0.005.

FIG. 3B. Specific killing activity was measured for ³⁵S labeledMDA-MB-231 breast cancer cells that were incubated with NK cells fromhealthy donor (at ratio of 1:10) and with control mIgG (left bar) oranti TIGIT mAb VSIG9#1 (right bar). **p<0.002.

FIG. 3C. Specific killing activity was measured for ³⁵S labeled Mel 562cells that were incubated with NK cells from healthy donor (at ratio of1:10) and with control mIgG (right bar), anti-TIGIT VSIG9#1 (left bar),or with anti TIGIT 258-CS1#4 (central bar). ** p<0.05.

FIG. 3D. Antibody-dependent cell-mediated cytotoxicity (ADCC) activitywas measured for ³⁵S labeled HepG2 cells that were incubated withanti-EGFR mAb (Erbitux®) and added to NK cells pre-incubated withcontrol mAb (left bar) or with anti-VSIG9#1 (right bar) (at ratio of10:1, Effectors:Target cells, respectively). **p<0.0007.

FIGS. 4A-4B. Antibodies 258-CS1#4 and VSIG9#1 block TIGIT binding toPVR. HepG2 cells that express the TIGIT ligand PVR were incubated withTIGIT-Fc or TIGIT-Fc following pre-incubation with mAb VSIG9#1 (FIG.4A), or with mAb 258-CS1#4 (FIG. 4B) and binding was measured usingFACS.

FIGS. 5A-5B. Binding kinetic analyses of the mAbs 258-cs1#4 (#4) (FIG.5A) and VSIG9#1 (FIG. 5B) to biosensor loaded with human TIGIT from twocommercial sources, using Biacore.

FIGS. 6A-6B. Antibody VSIG9#1 binds to TIGIT cells significantlystronger than commercial antibody MBSA43 (eBioscience Cat #12-9500-42).

FIG. 6A. FACS histogram plots of VSIG9#1 and the commercial antibodyMBSA43 binding to TIGIT. YTS-TIGIT cells (75*10³) were stained withanti-TIGIT antibodies VSIG9#1 (black open histogram) or MBSA43 (greyopen histogram) at a concentration range of from 250 nM to 65 fM inserial dilutions. Background—staining of mIgG (grey filled histograms).Each panel represents a specific concentration of the antibodies asindicated. Y-axis of each panel is cell counts and X-axis isfluorescence intensity (FL1-H).

FIG. 6B. Graphical analysis of the binding described in FIG. 6A. Half ofthe maximal intensity is achieved at the indicated points (MBSA43 greydashed, mAb VSIG#9#1 black dashed).

FIGS. 7A and 7B. The VSIG#9 antibody is significantly more potent inpreventing PVR-TIGIT interactions than the commercial antibody MBSA43 asmeasured by FACS analysis.

FIG. 7A. Histogram plots of YTS-TIGIT cells staining. 75*10³ YTS-TIGITcells were incubated with 2.5 pmole PVR-Fc (Grey open histograms) in thepresence of VSIG9#1 (Black open histograms) or MBSA43 (grey dashedhistograms) at a range of antibody concentrations from 27 to 0.014 pmolein a series of two fold dilutions. The bound PVR was detected by antiAlexa Fluor® 488 Mouse anti human IgG (BioLegend).

FIG. 7B. Histogram plots of YTS-TIGIT cells staining 75*10³ YTS-TIGITcells were incubated with 2.5 pmole PVR-Fc in the presence of VSIG9#1(black open histograms) or MBSA43 (Grey dashed histograms) at a range ofconcentrations from 27 to 0.014 pmole in a series of two fold dilutions.The bound anti TIGIT was detected by Alexa Fluor® 647 Goat anti-mouseIgG (BioLegend).

Each panel in FIGS. 7A and 7B represents a specific concentration of theantibodies as indicated. Y-axis of each panel is cell counts and X-axisis fluorescence intensity (FL1-H/FL-4). The numbers in the panelindicates Mean Fluorescent Intensity (MFI).

FIG. 8: Blocking of TIGIT by mAb VSIG9#1 (Vsig9.01) induces T cellproliferation alone or in combination with anti-PD-1 and anti-CTLA4.PBMCs from healthy donors were labeled with 5 (6)-CarboxyfluoresceinN-hydroxysuccinimidyl ester (CFSE) and activated by anti-CD3 antibodiesfollowed by incubation with MDA-MB-231 cells over expressing hCD80 inpresence of 4 μg/ml of the indicated mAbs for 5-9 days. Proliferationwas measured by CFSE dilutions. Representative data from at least 5different experiments is shown. *p<0.04 **<0.015 ***<0.0004.

FIG. 9: Anti-TIGIT VSIG9#1 mAb synergize with anti PD-1 and anti CTLA-4in increasing CD8 T cells in AML cells. Immune cells separated from bonemarrow aspirate obtained from an AML patient were co-cultured with 4μg/ml of anti TIGIT, anti PD-1 and/or anti CTLA-4 antibodies for 12 dayand the amount of CD8 T cells were then determined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies specific to thehuman protein TIGIT, some of the mAbs having exceptional high affinityto this protein and some have dual specificity, and can bind differentdomains of the protein. The invention also provides production and useof the mAbs as therapeutic agents. In particular, the mAbs of thepresent invention may be used, alone or in combination with otheragents, for restoring and augmenting anti-tumor killing activity of NKand other cells, and as diagnostic reagents.

The term “antigen” as used herein refers to a molecule or a portion of amolecule capable of eliciting antibody formation and being specificallybound by an antibody. An antigen may have one or more than one epitope.The specific binding referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies which may beevoked by other antigens. An antigen according to some embodiments ofthe present invention is a TIGIT protein, having an accession numberselected from the group consisting of: NP_776160.2; Q495A1.1;AAI01290.1; AAI01291.1; AAI01292.1; ACD74757.1; EAW79602.1; andAIC53385.1; or a fragment of any of said TIGIT proteins.

According to some embodiments the monoclonal antibodies of the presentinvention are specific to human TIGIT. According to yet otherembodiments, mAb according to the invention bind human TIGIT and atleast one TIGIT from other species, such as mouse, monkey, dog orothers. According to some specific embodiments, mAbs bind to human TIGITand to at least one TIGIT monkey species.

The term “antigenic determinant” or “epitope” as used herein refers tothe region of an antigen molecule that specifically reacts with aparticular antibody. Peptide sequences derived from an epitope can beused, alone or in conjunction with a carrier moiety, applying methodsknown in the art, to immunize animals and to produce additionalpolyclonal or monoclonal antibodies. Isolated peptides derived from anepitope may be used in diagnostic methods to detect antibodies.

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulfide bonds and two light chains, each light chain beinglinked to a respective heavy chain by disulfide bonds in a “Y” shapedconfiguration. Proteolytic digestion of an antibody yields Fv (Fragmentvariable) and Fc (Fragment crystalline) domains. The antigen bindingdomains, Fab, include regions where the polypeptide sequence varies. Theterm F(ab′)₂ represents two Fab′ arms linked together by disulfidebonds. The central axis of the antibody is termed the Fc fragment. Eachheavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains (C_(H)). Each light chain has a variabledomain (V_(L)) at one end and a constant domain (C_(L)) at its otherend, the light chain variable domain being aligned with the variabledomain of the heavy chain and the light chain constant domain beingaligned with the first constant domain of the heavy chain (CH1). Thevariable domains of each pair of light and heavy chains form theantigen-binding site. The domains on the light and heavy chains have thesame general structure and each domain comprises four framework regions,whose sequences are relatively conserved, joined by three hyper-variabledomains known as complementarity determining regions (CDRs 1-3). Thesedomains contribute specificity and affinity of the antigen-binding site.

CDR determination—CDR identification from a given heavy or light chainvariable sequence, is typically made using one of few methods known inthe art. For example, such determination is made according to the Kabat(Wu T. T and Kabat E. A., J Exp Med, 1970; 132:211-50) and IMGT (LefrancM-P, et al., Dev Comp Immunol, 2003, 27:55-77).

When the term “CDR having a sequence”, or a similar term is used, itincludes options wherein the CDR comprises the specified sequences andalso options wherein the CDR consists of the specified sequence.

The antigen specificity of an antibody is based on the hypervariableregions, namely the unique CDR sequences of both light and heavy chainsthat together form the antigen-binding site.

The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu)determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,respectively). The light chain is either of two isotypes (kappa, κ orlambda, λ) found in all antibody classes.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies,multi-specific antibodies (e.g., bi-specific antibodies), and antibodyfragments long enough to exhibit the desired biological activity, namelybinding to human TIGIT.

Antibody or antibodies according to the invention include intactantibodies, such as polyclonal antibodies or monoclonal antibodies(mAbs), as well as proteolytic fragments thereof, such as the Fab orF(ab′)₂ fragments. Single chain antibodies also fall within the scope ofthe present invention.

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CHI domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) whichconsists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)₂fragments, a bivalent fragment including two Fab′ fragments linked by adisulphide bridge at the hinge region; (ix) single chain antibodymolecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242,423-426; and Huston et al., PNAS (USA) 1988, 85, 5879-5883); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; andHollinger et at, Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi)“linear antibodies” comprising a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).

Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al, Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv).

Single chain antibodies can be single chain composite polypeptideshaving antigen binding capabilities and comprising amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain i.e. linked V_(H)-V_(L) or single chain Fv (scFv).Techniques for the production of single-chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single-chain antibodies to TIGIT.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” is not to be construed as requiring production ofthe antibody by any particular method. mAbs may be obtained by methodsknown to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature1975, 256, 495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described, forexample, in Clackson et al., Nature 1991, 352, 624-628 or Marks et al.,J. Mol. Biol., 1991, 222:581-597.

The design and development of recombinant monovalent antigen-bindingmolecules derived from monoclonal antibodies through rapididentification and cloning of the functional variable heavy (VH) andvariable light (VL) genes and the design and cloning of a synthetic DNAsequence optimized for expression in recombinant bacteria are describedin Fields et at. 2013, 8(6):1125-48.

The mAbs of the present invention may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA. A hybridoma producing a mAb may becultivated in-vitro or in-vivo. High titers of mAbs can be obtained byin-vivo production where cells from the individual hybridomas areinjected intra-peritoneally into pristine-primed Balb/c mice to produceascites fluid containing high concentrations of the desired mAbs. mAbsof isotype IgM or IgG may be purified from such ascites fluids, or fromculture supernatants, using column chromatography methods well known tothose of skill in the art.

Besides the conventional method of raising antibodies in vivo,antibodies can be generated in vitro using phage display technology.Such a production of recombinant antibodies is much faster compared toconventional antibody production and they can be generated against anenormous number of antigens. Furthermore, when using the conventionalmethod, many antigens prove to be non-immunogenic or extremely toxic,and therefore cannot be used to generate antibodies in animals.Moreover, affinity maturation (i.e., increasing the affinity andspecificity) of recombinant antibodies is very simple and relativelyfast. Finally, large numbers of different antibodies against a specificantigen can be generated in one selection procedure. To generaterecombinant mAbs one can use various methods all based on displaylibraries to generate a large pool of antibodies with different antigenrecognition sites. Such a library can be made in several ways: One cangenerate a synthetic repertoire by cloning synthetic CDR regions in apool of H chain germline genes and thus generating a large antibodyrepertoire, from which recombinant antibody fragments with variousspecificities can be selected. One can use the lymphocyte pool of humansas starting material for the construction of an antibody library. It ispossible to construct naive repertoires of human IgM antibodies and thuscreate a human library of large diversity. This method has been widelyused successfully to select a large number of antibodies againstdifferent antigens. Protocols for bacteriophage library construction andselection of recombinant antibodies are provided, for example in CurrentProtocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc.(1992-2000), Chapter 17, Section 17.1.

Non-human antibodies may be humanized by any methods known in the art.In one method, the non-human CDRs are inserted into a human antibody orconsensus antibody FR sequence. Further changes can then be introducedinto the antibody framework to modulate affinity or immunogenicity.

For example, U.S. Pat. No. 5,585,089 of Queen et al. discloses ahumanized immunoglobulin and methods of preparing same, wherein thehumanized immunoglobulin comprises CDRs from a donor immunoglobulin andV_(H) and V_(L) region FRs from human acceptor immunoglobulin H and Lchains, wherein said humanized immunoglobulin comprises amino acids fromthe donor immunoglobulin FR outside the Kabat and Chothia CDRs, andwherein the donor amino acids replace corresponding amino acids in theacceptor immunoglobulin H or L chain frameworks.

Also, transgenic mice, or other organisms such as other mammals, can beused to express humanized antibodies.

U.S. Pat. No. 5,225,539, of Winter, also discloses an altered antibodyor antigen-binding fragment thereof and methods of preparing same,wherein a V domain of the antibody or antigen-binding fragment has theFRs of a first immunoglobulin H or L chain V domain and the CDRs of asecond immunoglobulin V_(H) or V_(L) domain, wherein said secondimmunoglobulin V_(H) or V_(L) domain is different from said firstimmunoglobulin V_(H) or V_(L) domain in antigen binding specificity,antigen binding affinity, stability, species, class or subclass.

Anti-idiotype antibodies specifically immunoreactive with thehypervariable regions of an antibody of the invention are alsocomprehended.

Alternatively, phage display technology can be utilized to selectantibody genes with binding activities towards human TIGIT either fromrepertoires of PCR amplified v-genes of lymphocytes from humans screenedfor possessing anti-VEGF or from libraries (McCafferty, et al., (1990),Nature 348, 552-554; Marks, et al., (1992) Biotechnology 10, 779-783).The affinity of these antibodies can also be improved by, for example,chain shuffling (Clackson et al., (1991) Nature 352:628).

The above-described antibodies can be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides by, forexample, affinity chromatography.

The invention provides a monoclonal antibody or an antibody fragmentcomprising an antigen binding domain (ABD) which comprises three CDRs ofa light chain and three CDRs of a heavy chain, wherein said ABD has atleast 90% sequence identity or similarity with an ABD of a monoclonalmouse antibody comprising: (i) a heavy variable chain comprising theamino acid sequence SEQ ID NO:7 and a light variable chain comprisingthe amino acid sequence SEQ ID NO:8 (herein identified as VSIG9#1); or aheavy variable chain comprising the amino acid sequence SEQ ID NO:18 anda light variable chain comprising the amino acid sequence SEQ ID NO:19(herein identified as 258-CS1#4 (or #4)). Such antibody may have an ABDdomain having at least 93%, at least 94%, at least 95%, at least 96, atleast 97, at least 98, at least 99% sequence identity or similarity or100% sequence identity with corresponding ABD of VSIG9#1 or 258-CS1#4.

Sequence identity is the amount of amino acids or nucleotides whichmatch exactly between two different sequences. Sequence similaritypermits conservative substitution of amino acids to be determined asidentical amino acids.

The invention also provides conservative amino acid variants of theantibody molecules according to the invention. Variants according to theinvention also may be made that conserve the overall molecular structureof the encoded proteins. Given the properties of the individual aminoacids comprising the disclosed protein products, some rationalsubstitutions will be recognized by the skilled worker. Amino acidsubstitutions, i.e. “conservative substitutions,” may be made, forinstance, on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. The term “antibody analog” as used herein refers toan antibody derived from another antibody by one or more conservativeamino acid substitutions.

The term “antibody variant” as used herein refers to any moleculecomprising the antibody of the present invention. For example, fusionproteins in which the antibody or an antigen-binding-fragment thereof islinked to another chemical entity is considered an antibody variant.

Analogs and variants of the antibody sequences are also within the scopeof the present application. These include but are not limited toconservative and non-conservative substitution, insertion and deletionof amino acids within the sequence. Such modification and the resultantantibody analog or variant are within the scope of the present inventionas long as they confer, or even improve the binding of the antibody tothe human TIGIT.

Conservative substitutions of amino acids as known to those skilled inthe art are within the scope of the present invention. Conservativeamino acid substitutions include replacement of one amino acid withanother having the same type of functional group or side chain, e.g.,aliphatic, aromatic, positively charged, negatively charged. Thesesubstitutions may enhance oral bioavailability, penetration into theislets, targeting to specific beta cell populations, immunogenicity, andthe like. One of skill will recognize that individual substitutions,deletions or additions to a peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” where the alteration results in the substitution of an aminoacid with a chemically similar amino acid. Conservative substitutiontables providing functionally similar amino acids are well known in theart. For example, according to one table known in the art, the followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “human antibody” as used herein refers to an antibody whichpossesses an amino acid sequence which corresponds to that of anantibody produced by a human and/or has been made using any of thetechniques for making human antibodies as disclosed herein. Thisdefinition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues. Human antibodiescan be produced using various techniques known in the art.

The terms “molecule having the antigen-binding portion of an antibody”and “antigen-binding-fragments” as used herein is intended to includenot only intact immunoglobulin molecules of any isotype and generated byany animal cell line or microorganism, but also the antigen-bindingreactive fraction thereof, including, but not limited to, the Fabfragment, the Fab′ fragment, the F(ab)₂ fragment, the variable portionof the heavy and/or light chains thereof, Fab mini-antibodies (see e.g.,WO 93/15210, U.S. patent application Ser. No. 08/256,790, WO 96/13583,U.S. patent application Ser. No. 08/817,788, WO 96/37621, U.S. patentapplication Ser. No. 08/999,554), dimeric bispecific mini-antibodies(see Muller et al., FEBS Lett. 1998 Jul. 31; 432(1-2):45-9) andsingle-chain antibodies incorporating such reactive fraction, as well asany other type of molecule in which such antibody reactive fraction hasbeen physically inserted. Such molecules may be provided by any knowntechnique, including, but not limited to, enzymatic cleavage, peptidesynthesis or recombinant techniques.

The term “non-fully-humanized monoclonal antibody” as used herein refersto a monoclonal antibody, having a heavy chain and/or a light chainvariable domains in which the amino-acid sequences flanking and/orimmediately adjacent to the CDRs are not fully human, i.e. are notidentical to any known homologous or corresponding sequences taken fromnatural human antibodies.

Humanized and Human Antibodies

A humanized antibody, typically has a human FR grafted with non-humanCDRs. Thus, a humanized antibody has one or more amino acid sequenceintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human V domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human V_(H) and V_(L) domains to be used in making thehumanized antibodies is very important for reducing immunogenicity.According to the so-called “best-fit” method, the sequence of the Vdomain of a rodent antibody is screened against the entire library ofknown human-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human FR for the humanizedantibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al, J.Mol. Biol., 196:901 (1987)). Another method uses a particular FR derivedfrom the consensus sequence of all human antibodies of a particularsubgroup of H or L chains. The same FR may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et at, J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh specificity and affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

Pharmacology

In pharmaceutical and medicament formulations, the active agent ispreferably utilized together with one or more pharmaceuticallyacceptable carrier(s) and optionally any other therapeutic ingredients.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The active agent is provided in anamount effective to achieve the desired pharmacological effect, asdescribed above, and in a quantity appropriate to achieve the desireddaily dose.

Typically, the antibodies and fragments and conjugates thereof of thepresent invention comprising the antigen binding portion of an antibodyor comprising another polypeptide including a peptide-mimetic will besuspended in a sterile saline solution for therapeutic uses. Thepharmaceutical compositions may alternatively be formulated to controlrelease of active ingredient (molecule comprising the antigen bindingportion of an antibody) or to prolong its presence in a patient'ssystem. Numerous suitable drug delivery systems are known and include,e.g., implantable drug release systems, hydrogels,hydroxymethylcellulose, microcapsules, liposomes, microemulsions,microspheres, and the like. Controlled release preparations can beprepared through the use of polymers to complex or adsorb the moleculeaccording to the present invention. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebaric acid. Therate of release of the molecule according to the present invention,i.e., of an antibody or antibody fragment, from such a matrix dependsupon the molecular weight of the molecule, the amount of the moleculewithin the matrix, and the size of dispersed particles.

The pharmaceutical composition of this invention may be administered byany suitable means, such as orally, topically, intranasally,subcutaneously, intramuscularly, intravenously, intra-arterially,intraarticulary, intralesionally or parenterally. Ordinarily,intravenous (i.v.) administration is used for delivering antibodies.

It will be apparent to those of ordinary skill in the art that thetherapeutically effective amount of the molecule according to thepresent invention will depend, inter alia upon the administrationschedule, the unit dose of molecule administered, whether the moleculeis administered in combination with other therapeutic agents, the immunestatus and health of the patient, the therapeutic activity of themolecule administered and the judgment of the treating physician. Asused herein, a “therapeutically effective amount” refers to the amountof a molecule required to alleviate one or more symptoms associated witha disorder being treated over a period of time.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the durationof survival, time to disease progression (TTP), the response rates (RR),duration of response, and/or quality of life.

The cancer amendable for treatment by the present invention includes,but is not limited to: carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high-grade immunoblastic NHL; high-gradelymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting of breastcancer, colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, livercancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. The cancerous conditions amendablefor treatment of the invention include metastatic cancers.

The molecules of the present invention as active ingredients aredissolved, dispersed or admixed in an excipient that is pharmaceuticallyacceptable and compatible with the active ingredient as is well known.Suitable excipients are, for example, water, saline, phosphate bufferedsaline (PBS), dextrose, glycerol, ethanol, or the like and combinationsthereof. Other suitable carriers are well known to those skilled in theart. In addition, if desired, the composition can contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents.

The pharmaceutical composition according to the present invention may beadministered together with an anti-neoplastic composition.

The term “Treatment” as used herein refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include melanoma, lung, thyroid,breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic,leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, orendometrial cancer.

According to some embodiments, the method of treating cancer comprisesadministering the pharmaceutical composition as part of a treatmentregimen comprising administration of at least one additional anti-canceragent.

According to some embodiments, the anti-cancer agent is selected fromthe group consisting of an antimetabolite, a mitotic inhibitor, ataxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, anasparaginase, an alkylating agent, an antitumor antibiotic, andcombinations thereof. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the antimetabolite is selected from thegroup consisting of cytarabine, gludarabine, fluorouracil,mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea.According to some embodiments, the mitotic inhibitor is selected fromthe group consisting of vincristine, vinblastine, and vinorelbine.According to some embodiments, the topoisomerase inhibitor is selectedfrom the group consisting of topotecan and irenotecan. According to someembodiments, the alkylating agent is selected from the group consistingof busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide,cisplatin, carboplatin, ifosamide, mechlorethamine, melphalan, thiotepa,dacarbazine, and procarbazine. According to some embodiments, theantitumor antibiotic is selected from the group consisting of bleomycin,dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin,mitoxantrone, and plicamycin. According to some embodiments, thetopoisomerase II is selected from the group consisting of etoposide andteniposide. Each possibility represents a separate embodiment of thepresent invention.

According to some particular embodiments, the anti-cancer agent isselected from the group consisting of bevacizumab, carboplatin,cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride,topotecan hydrochloride, thiotepa, and combinations thereof. Eachpossibility represents a separate embodiment of the present invention.

Monoclonal antibodies according to the present invention may be used aspart of combined therapy with at least one anti-cancer agent. Accordingto some embodiments, the additional anti-cancer agent is animmuno-modulator, an activated lymphocyte cell, a kinase inhibitor or achemotherapeutic agent.

According to some embodiments, the anti-cancer agent is animmuno-modulator, whether agonist or antagonist, such as antibodyagainst a checkpoint molecule.

Checkpoint immunotherapy blockade has proven to be an exciting new venueof cancer treatment. Immune checkpoint pathways consist of a range ofco-stimulatory and inhibitory molecules which work in concert in orderto maintain self-tolerance and protect tissues from damage by the immunesystem under physiological conditions. Tumors take advantage of certaincheckpoint pathways in order to evade the immune system. Therefore, theinhibition of such pathways has emerged as a promising anti-cancertreatment strategy.

The anti-cytotoxic T lymphocyte 4 (CTLA-4) antibody ipilimumab (approvedin 2011) was the first immunotherapeutic agent that showed a benefit forthe treatment of cancer patients. The antibody interferes withinhibitory signals during antigen presentation to T cells.Anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in2014) blocks negative immune regulatory signaling of the PD-1 receptorexpressed by T cells. An additional anti-PD-1 agent was filed forregulatory approval in 2014 for the treatment of non-small cell lungcancer (NSCLC). Active research is currently exploring many other immunecheckpoints, among them: CEACAM1, lymphocyte activation gene 3 (LAG3),CD137, OX40 (also referred to as CD134), and killer cellimmunoglobulin-like receptors (KIR).

According to some specific embodiments, the immuno-modulator is selectedfrom the group consisting of: an antibody inhibiting CTLA-4, ananti-human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2antibody, an activated cytotoxic lymphocyte cell, a lymphocyteactivating agent, an antibody against CEACAM, and a RAF/MEK pathwayinhibitor. Each possibility represents a separate embodiment of thepresent invention. According to some specific embodiments, theadditional immuno-modulator is selected from mAb to PD-1, mAb to PD-L1,mAb to PD-L2, mAb to CEACAM1, mAb to CTLA-4, Interleukin 2 (IL-2) orlymphokine-activated killer (LAK) cell.

According to other embodiments the anti-cancer agent is achemotherapeutic agent. The chemotherapy agent, which could beadministered together with the antibody according to the presentinvention, or separately, may comprise any such agent known in the artexhibiting anticancer activity, including but not limited to:mitoxantrone, topoisomerase inhibitors, spindle poison vincas:vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel;alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide,melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil,cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan,topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin,mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin,idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin;interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide,and megestrol acetate.

According to some embodiments, the chemotherapeutic agent is selectedfrom alkylating agents, antimetabolites, folic acid analogs, pyrimidineanalogs, purine analogs and related inhibitors, vinca alkaloids,epipodophyllotoxins, antibiotics, L-asparaginase, topoisomeraseinhibitor, interferons, platinum coordination complexes, anthracenedionesubstituted urea, methyl hydrazine derivatives, adrenocorticalsuppressant, adrenocorticosteroides, progestins, estrogens,antiestrogen, androgens, antiandrogen, and gonadotropin-releasinghormone analog. According to another embodiment, the chemotherapeuticagent is selected from the group consisting of 5-fluorouracil (5-FU),leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel anddoxetaxel. One or more chemotherapeutic agents can be used.

In some embodiments the pharmaceutical composition according to thepresent invention is for use in treating cancer or for use in enhancingthe immune response.

The term “enhancing immune response” refers to increasing theresponsiveness of the immune system and prolonging its memory. Thepharmaceutical composition according to the present invention may beused to stimulate immune system upon vaccination. Thus in one embodimentthe pharmaceutical composition can be used for improving vaccination.

In certain embodiments, the cancer is selected from lung, thyroid,breast, colon, melanoma, prostate, hepatic, bladder, renal, cervical,pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma,biliary, and endometrial cells cancer. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, a pharmaceutical composition, comprisingat least one antibody or fragment thereof according to the presentinvention, and a pharmaceutical composition, comprising an additionalimmuno-modulator or a kinase inhibitor, are used in treatment of cancerby separate administration.

According to still another aspect the present invention provides amethod of treating cancer in a subject in need thereof comprisingadministering to said subject a therapeutically effective amount of amonoclonal antibody or antibody fragment according to the presentinvention.

The term “treating” refers to taking steps to obtain beneficial ordesired results, including clinical results. Beneficial or desiredclinical results include, but are not limited to, alleviation oramelioration of one or more symptoms associated muscular dystrophy,delay or slowing of that impairment, amelioration, palliation orstabilization of that impairment, and other beneficial results.

The term “effective amount” as used herein refers to a sufficient amountof the monoclonal antibody of the antibody fragment that, whenadministered to a subject will have the intended therapeutic effect. Theeffective amount required to achieve the therapeutic end result maydepend on a number of factors including, for example, the specific typeof the tumor and the severity of the patient's condition, and whetherthe combination is further co-administered with radiation. The effectiveamount (dose) of the active agents, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the subject over time, including but not limited toinhibition of tumor growth, reduction in the rate of tumor growth,prevention of tumor and metastasis growth and enhanced survival.

Toxicity and therapeutic efficacy of the compositions described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., by determining the IC50 (theconcentration which provides 50% inhibition) and the maximal tolerateddose for a subject compound. The data obtained from these cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. The dosage may vary depending inter alia upon thedosage form employed, the dosing regimen chosen, the composition of theagents used for the treatment and the route of administration utilizedamong other relevant factors. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. Depending on the severity andresponsiveness of the condition to be treated, dosing can also be asingle administration of a slow release composition, with course oftreatment lasting from several days to several weeks or until cure iseffected or diminution of the disease state is achieved. The amount of acomposition to be administered will, of course, be dependent on thesubject being treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, and all otherrelevant factors.

The term “administering” or “administration of” a substance, a compoundor an agent to a subject can be carried out using one of a variety ofmethods known to those skilled in the art. For example, a compound or anagent can be administered enterally or parenterally. Enterally refers toadministration via the gastrointestinal tract including per os,sublingually or rectally. Parenteral administration includesadministration intravenously, intradermally, intramuscularly,intraperitoneally, subcutaneously, occularly, sublingually,intranasally, by inhalation, intraspinally, intracerebrally, andtransdermally (by absorption, e.g., through a skin duct). A compound oragent can also appropriately be introduced by rechargeable orbiodegradable polymeric devices or other devices, patches and pumps, orformulations, which provide for the extended, slow or controlled releaseof the compound or agent. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods. In some embodiments, the administration includes both directadministration, including self-administration, and indirectadministration, including the act of prescribing a drug. For example, asused herein, a physician who instructs a patient to self-administer adrug, or to have the drug administered by another and/or who provides apatient with a prescription for a drug is administering the drug to thepatient.

Antibodies are generally administered in the range of about 0.1 to about20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, andoften about 1 to about 5 mg/k. In this regard, it is preferred to useantibodies having a circulating half-life of at least 12 hours,preferably at least 4 days, more preferably up to 21 days. Chimeric andhumanized antibodies are expected to have circulatory half-lives of upto four and up to 14-21 days, respectively. In some cases it may beadvantageous to administer a large loading dose followed by periodic(e.g., weekly) maintenance doses over the treatment period. Antibodiescan also be delivered by slow-release delivery systems, pumps, and otherknown delivery systems for continuous infusion.

The term “about” means that an acceptable error range, e.g., up to 5% or10%, for the particular value should be assumed.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES

Experimental Procedures

NK cell cytotoxicity of various tumors is inhibited due to ligandbinding to the TIGIT protein present on all human NK cells and onvarious T cells. Anti-hTIGIT mAbs were generated and tested for theirability to antagonize killing inhibition imposed by ligand interactionwith hTIGIT.

The following cell lines were used: the human EBV transformed 721.221cells, the human NK tumor cell line YTS ECO, MEL562 melanoma cells,MDA-MB-231 breast cancer cells and HepG2 human hepatocellular cells. Thegeneration of the various YTS ECO transfectants: YTS hTIGIT wasdescribed previously (Stanietsky et al., 2009). All cells were grown inRPMI medium supplemented with 10% FCS.

For killing assays, target cells were grown overnight in the presence of³⁵S-Methionine added to a methionine-free media (Sigma). Prior toincubation with the effectors cells (NK cells), cells were washed,counted, and 5000 cells/well were plated. 0.5 μg of mAb of blockingantibody was used. For each target, the spontaneous ³⁵S release wascalculated using cells, which were not incubated with effector cells,and maximum [³⁵S]-release was calculated by applying 100 μl 0.1 M NaOHto the target cells. The amount of [³⁵S]-release was measured after 5hours of incubation with effectors (at 37° C.) by a β-counter MicroBeta²(PerkinElmer).

K_(D) Determination Using Biacore

Surface plasmon resonance (SPR) Biosensor Biacore™ T100 (GE Healthcare)was used to determine Koff, Kon and K_(D) between the antibodies andTIGIT.

Example 1. Production of Monoclonal Antibodies Specific to TIGIT

Monoclonal antibodies against human TIGIT were generated according toone example, by immunizing with TIGIT-Fc fusion protein. The codingsequence of human TIGIT was produced by cloning as a fusion to the Fcfragment of IgG1. The recombinant fusion protein generated was injectedto mice and hybridoma supernatants were tested for specific recognitionof YTS NK cell line transfectants expressing TIGIT.

Total RNA was extracted from results hybridoma cells following thetechnical manual of TRIzol® Reagent (Ambion, Cat. No.: 15596-026) andanalyzed by agarose gel electrophoresis. Total RNA was reversetranscribed into cDNA using isotype-specific anti-sense primers oruniversal primers following the technical manual of PrimeScript™ 1stStrand cDNA Synthesis Kit (Takara, Cat. No.: 6110A). The antibodyfragments of VH, VL, CH and CL were amplified according to a standardoperating procedure of rapid amplification of cDNA ends (RACE).Amplified antibody fragments were separately cloned into a standardcloning vector using standard molecular cloning procedures.

Colony PCR screening was performed to identify clones with inserts ofcorrect sizes. No less than five single colonies with inserts of correctsizes were sequenced for each antibody fragment.

Results

Two exemplary monoclonal antibodies specific to human TIGIT producedwere termed #4 (or 258-cs1#4) and VSIG9#1 (or Vsig9.01). These mAbsrecognizes YTS cells transfected with human TIGIT as demonstrated inFIG. 1A.

The isolated total RNA of the sample was run alongside a DNA markerMarker III (TIAGEN, Cat No.: MD103) on a 1.5% agarose/GelRed™ gel.

Four microliters of PCR products of each of sample were run alongsidethe DNA Marker III on 1.5% agarose/GelRed™ gel. The PCR products werepurified and stored at −20° C. until further use.

The VH, VL of different clones were sequenced. The sequences of thevariable regions, listed below, are of the antibodies produced by twohybridoma clones termed VSIG9#1 and 258-cs1.04. The CDR sequences ineach amino acid chain are underlined.

Antibody VSIG9 #1 Heavy chain: DNA sequence (SEQ ID NO: 9)CAGGTGCAGCTGCAGGAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTATGGTATAAGCTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAGATTTATCCCAGAAGTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCGTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAAAGGGACCCTACTATACTAAGAACGAGGACTACTGGGGCCAAGGCACCATTCTCACAGTCTCCTCAAntibody VSIG9 #1 Heavy chain: Amino acids sequence (SEQ ID NO: 7)QVQLQESGAELARPGASVKLSCKAS GYTFTSYGIS WVKQRTGQGLEWIG EIYPRS GNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCAR KGPYYTKNEDY WGQGTILTVSS.Antibody VSIG9 #1 Light chain: DNA sequence (SEQ ID NO: 10)GACATCCAGATGACTCAGTCTCCAGCCTCCCTGGCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGCACATTTACTACAGTTTAGCATGGTATCAGCAGAAGCAAGGGAAATCTCCTCAGCTCCTGATCTATAATGCAAACAGCTTGGAAGATGGTGTCCCATCGAGGTTCAGTGGCAGTGGATCTGGGACACAATATTCTATGAAGATCAACAGCATGCAGCCTGAAGATACCGCAACTTATTTCTGTAAACAGGCTTATGACGTTCCTCGGACCTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGG CTGATGCTGCACCAACTGTATCC.Antibody VSIG9 #1 HLight chain: Amino acids sequence (SEQ ID NO: 8)DIQMTQSPASLAASVGETVTITC RASEHIYYSLA WYQQKQGKSPQLLIY NANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFC KQAYDVPRT FGGGTKLEIKRADA APTVSAntibody 258-cs1.04 (also denoted #4)Antibody 258-cs1.04 Heavy chain: DNA sequence (SEQ ID NO: 20)CAGGTCCAACTGCTGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCATCTACTGTATACACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAGTCCTAGCAACGGTCGTACTATCTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTATAGACAAATCCTCCACCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTGCTGTGCAATATCGGATGGTTACGACGGATACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.Antibody 258-cs1.04_Heayy chain: Amino acids sequence (SEQ ID NO: 18)QVQLLQPGAELVKPGASVKLSCKASGYTFT IYCIH WVKQRPGQGLEWIG EISPSNG RTIYNEKFKNKATLTIDKSSTTAYMQLSSLTSEDSAVYCCAI SDGYDGYYFDY WG QGTTLTVSS.Antibody 258-cs1.04_Light chain: DNA sequence (SEQ ID NO: 21)GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAACTGGCTTCAGCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTAGATTCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGACTTGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAA .TATGCTAGTTATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAAntibody 258-cs1.04_Light chain: Amino acids sequence (SEQ ID NO: 19)DIQMTQSPSSLSASLGERVSLTC RAS Q EISGYLN WLQQKPDGTIKRLIYA ASTLDS GVPKRFSGSRSGSDYSLTISRLESEDFADYYC LQYASYPRT FGGGTKLEIK.Table 1 lists the CDR and variable region amino acid sequences of thetwo anti human TIGIT mAb and the variable region sequences.

TABLE 1  SEQ SEQ ID ID CDR VSIG9 #1 No. 258-cs1.04 No. HCDR1 GYTFTSYGIS1 IYCIH 12 HCDR2 EIYPRSGNTYYNEKFKG 2 EISPSNGRTIYNEKFKN 13 HCDR3KGPYYTKNEDY 3 SDGYDGYYFDY 14 LCDR1 RASEHIYYSLA 4 RASQEISGYLN 15 LCDR2NANSLED 5 AASTLDS 16 LCDR3 KQAYDVPRT 6 LQYASYPRT 17 VHAmino acid sequence 7 Amino acid sequence 18 VH Polynucleotide sequence9 Polynucleotide sequence 20 VL Amino acid sequence 8Amino acid sequence 19 VL Polynucleotide sequence 10Polynucleotide sequence 21

Example 2. Broad Expression of TIGIT on Effector Immune Cells

To examine the recognition of TIGIT by the mAbs, 2*10⁵ YTS-TIGIT overexpressing cells (as previously described by Stanietsky et al., ibid)were incubated with 0.2 microgram of anti TIGIT mAb clone VSIG9#1 or #4on ice for 30 min. After two rounds of wash in FACS buffer, goat-antimouse IgG (H+L) Secondary Antibody Alexa Fluor® 647 conjugate(BioLegend) was added for additional 30 min on ice. As shown in FIG. 1A,the mAbs recognize TIGIT proteins on YTS-TIGIT over expressing cells(right curve), but not on YTS cells. mIgG was used as a control. Next,the VSIG9#1 was examined on activated NK cells from two healthy donors.As shown in FIG. 1B, the mAb recognized the NK cells (right curve in theFACS histogram plots). mIgG was used as a control (left curve).

Next, the VSIG9#1 was examined on two CD8+ T cell populations, obtainedfrom melanoma patients (TIL-I and TIL-II). As shown in FIG. 1C, the mAbrecognized the T-cells (right curve in the FACS histogram plots). mIgGwas used as control (left curve). Overall, FIGS. 1A-1C shows broadexpression of TIGIT on immune cells and demonstrate that the VSIG9#1 mAbcan bind to immune cells.

Example 3. Anti-TIGIT VSIG9#1 Blocks TIGIT-Fc Binding to Tumor Cells

To examine the effect of VSIG9#1 on TIGIT-Fc binding to tumor cells,2.5*10⁵ HepG2 cells (that express high levels of PVR, Nectin-2 andNectin-3) were incubated with 25 microgram/well hTIGIT-Fc with no mAb(FIG. 2A, arrow 1) with mIgG (FIG. 2A, arrow 2) or withanti-TIGIT-VSIG9#1 at the indicated concentrations (FIG. 2A, arrows 3and 4) for 30 min on ice, the detection was done using Alexa Fluor® 647anti-human IgG (BioLegend). The expression of TIGIT ligands, PVR,Nectin-2 and Nectin-3 on the HepG2 cells is demonstrated in FIG. 2B.

The results demonstrate that the mAb can prevent TIGIT binding to tumorcells, suggesting that the antibody is capable of preventing theinhibition of the immune response.

Example 4. VSIG9#1 mAb Enhances Killing Activity

To examine the effect of anti-TIGIT mAbs on the killing activity ofeffector cells, YTS-TIGIT NK cells were incubated with ³⁵S labeled721.221-PVR cells in the presence of 2.5 microgram/ml of control mIgG(left bar) or anti-TIGIT VSIG9#1 (right bar)(*p<0.02). The specifickilling was calculated as previously described (Stanietsky et. al ibid).The killing percentage was examined after 5 hours. As shown in FIG. 3A,anti TIGIT mAb VSIG9#1 enhances killing activity. Next, the specifickilling activity was examined for MDA-MB-231 human breast tumor cellsthat were labeled with ³⁵S and incubated with NK cells from healthydonors in the presence of control mIgG (left bar) or VSIG9#1 (rightbars). Killing percentage was measured after 5 hours (**p<0.002). Asshown in FIG. 3B, VSIG9#1 enhances the killing effect. The specifickilling activity of two antibodies was also examined with MEL562melanoma cells. As shown in FIG. 3C, the two mAbs, 258-CS1#4 (centralbar) and VSIG9#1 (left bar) significantly enhances (**p<0.05) thekilling effect of these cells compared to control antibody mIgG (rightbar). Finally, antibody-dependent cell-mediated cytotoxicity (ADCC) ofVSIG9#1 was examined on human hepatocellular HepG2 cells labeled with³⁵S that were incubated with anti-EGFR mAb (Erbitux®) and added to NKcells that were pre-incubated with control mAb (left bar) or withVSIG9#1 (right bar) at a ratio of 10:1 Effector cells:Target cells,respectively. (***p<0.0007). A shown in FIG. 3D, VSIG9#1 is capable ofenhancing the killing effect.

The results demonstrate that the tested anti human mAbs VSIG9#1 and258-CS1#4 are capable of enhancing the killing effect on differenttarget cells and in a variety of conditions. The mAbs prevent theinhibitory effect of the TIGIT receptor and are therefore suitable foruse as anti-cancer agents.

Example 5. Antibodies #4 and VSIG9#1 Block TIGIT Binding to PVR

To examine the ability of mAbs 258-CS1#4 and VSIG9#1 to block theinteraction of TIGIT which its ligands, HepG2 cells that express PVR,Nectin-2 and Nectin-3 were incubated with TIGIT-Fc or TIGIT-Fc followingpre-incubation with mAb VSIG-9#1 (FIG. 4A), or with mAb 258-CS1#4 (FIG.4B). The results showed that pre-incubation of TIGIT-Fc with VSIG-9 #1completely blocked TIGIT-Fc binding (FIG. 4A), while pre-incubation withmAB 258-CS1#4 partially blocked TIGIT-Fc binding (FIG. 4B).

Example 6. Antibodies 258-CS1#4 and VSIG-9#1 Exhibit High andExceptional Affinity to TIGIT

Full binding kinetic analyses of the mAbs to human TIGIT from twosources were carried out using Biacore. The results shown in FIGS. 5Aand 5B indicate that the two mAbs exhibit high affinity to human TIGIT.While the mAb termed 258-CS1#4 (#4) had an average Kd of about 1×10⁻⁷ M(average of 9.96×10⁻⁸ M and 1.07×10⁻⁷ M), mAb VSIG-9#1 binds human TIGITwith an extremely high affinity, having a Kd of 4.5×10⁻¹⁰ M (average of5.11⁻¹⁰ M and 3.87⁻¹⁰ M).

It was further demonstrated that mAb VSIG-9#1 is specific to human TIGITand does not bind mouse TIGIT.

Example 7. Antibody VSIG9#1 Exhibits Higher Affinity to TIGIT asCompared to the Commercial Antibody MBSA43

The binding affinity of antibodies VSIG9#1 and MBSA43 to TIGIT wascompared. 75×10³ TIGIT expressing YTS cells were stained with the twoantibodies at serial dilution concentrations of 250 nM to 65 fM,following determination by FACS. As shown in FIGS. 6A and 6B, VSIG#9stained the cells at a concentration lower as 62.5 fM, while MBSA43stained the cells only above 1950 fM (1.95 pM) and did not show stainingat the last 4 dilutions Clear staining differences between the twoantibodies are seen up to a concentration of 250 pM. The superiority ofthe VSIG9#1 antibody compared to the MBSA43 antibody is also depicted inFIG. 6B.

Example 8. The Blocking Activity of VSIG9#1 is Higher than the One ofthe Commercial Antibody MBSA43

To examine the anti-TIGIT antibodies blocking effect on PVR-TIGITbinding, a staining assay of TIGIT-expressing YTS cells with PVR-Fc wasperformed. 75×10³ YTS-TIGIT cells were incubated with 2.5 pmole PVR-Fcin the presence of VSIG9#1 or MBSA43 at a concentration range of from 27to 0.014 pmole in a series of two fold dilutions.

As shown in FIG. 7A (detection of bound PVR) and 7B (detection of boundanti TIGIT mAb), the PVR-TIGIT blocking activity of VSIG9#1 wassignificantly higher as compared to MBSA43.

The results of Examples 7 and 8 demonstrate that VSIG9#1 has significanthigher affinity to human TIGIT, as compared to MBSA43 and that it issignificantly better at preventing the binding of the high affinityligand (PVR) across a range of concentrations.

Example 9. Anti TIGIT mAb Synergistic Activity with Other CheckpointMolecule Inhibitors

The efficacy of mAb VSIG9#1 (Vsig9.01), alone or in combination withother immunomodulators, on induction of T cell proliferation by blockingof TIGIT, was examined. PBMCs from healthy donors were labeled with5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) and activatedwith anti-CD3 antibodies followed by incubation for 5-9 days withMDA-MB-231 cells over-expressing hCD80 in the presence of 4 μg/ml of themAb VSIG9#1, anti-PD-1 (Keytruda) and anti-CTLA4 (pilimumab), alone orin combination. Proliferation was measured by CFSE dilutions.

The results shown in FIG. 8, collected from at least 5 differentexperiments, indicate a synergism between the anti TIGIT mAb and antiPD-1 or anti CTLA4, demonstrated by significant increased T-cellproliferation for the combination of VSIG9#1 with the other checkpointantibodies (*p<0.04 **<0.015 ***<0.0004).

Example 10. In Vivo Effect of the Humanized Anti-TIGIT Antibodies in aMouse Model of Human Tumor

The anti-tumor efficacy of the antibodies is studied in vivo. Toestimate the efficacy of the antibodies described herein in inhibitionof human cancer, the antibody is studied in a model combining bothtumors and lymphocytes of human origin. Severe combined immune-deficientmice (SCID) is engrafted with hPBL to restore immune-competence. Miceare challenged with human cancer cells and treated with increasingconcentrations of the anti human TIGIT antibody, administered in single-or multi-intravascular dose several days post tumor challenge.

A similar model with tumor lines in SCID mice is performed according toPaine-Murrieta G D, Cancer Chemother Pharmacol. 1997; 40(3):209-14.

Example 11. Inhibition of Human Melanoma (SK-28) in SCID Mice by theHumanized Anti-TIGIT Antibodies

To estimate the efficacy of anti-TIGIT antibodies in inhibition of humancancer, the modified antibody is studied in a model combining bothtumors and lymphocytes of human origin. Severe combined immune-deficientmice (SCID) is engrafted with hPBL to restore immune-competence. Miceare challenged with human melanoma cells (SK-28) and treated withincreasing concentrations of the antibody, administered in a single i.v.dose on day 11 post tumor inoculation.

Similarly, a model described by Hardy el at, Proc Natl Acad Sci USA.1997 May 27; 94(11): 5756-5760 is employed.

Example 12. Immunotherapy of Human Colorectal Cancer Hepatic Metastasesby the Anti-TIGIT mAbs in Nude Mice

LIME and HM7 are two sub-clones of the human CRC cell line LS174T thatwere selected for their high mucin synthesis and metastatic potential.The tumor cells are injected into the exposed spleen of anesthetizednude mice. After several minutes, the spleens are removed and theexcisions closed. Low doses of anti-TIGIT antibodies of the inventionare administered 10 days later and mice are sacrificed 35 days posttumor inoculation. The livers are weighed, the number of metastaticnodules counted, and liver tissue is processed for histology andImmunohistochemistry study.

Additional metastasis models that can be used to test the antibodies andfragments of the present invention were described by Yung at al., OculOncol Pathol. 2015 April; 1(3): 151-160.

Example 13. Anti TIGIT Antibodies have Synergistic Effect on AML Cells

Bone marrow aspirate was obtained from an AML patient, following Ficollseparation the immune cells and the blasts were co-cultured with variousantibodies (at 4 μg/ml) for 12 days after 12 days the amount of T cellswas established.

As demonstrated in FIG. 9, significant increase in the amount of CD8 Tcells was observed in presence of anti-TIGIT VSIG9#1 mAb. Interestinglyblocking of TIGIT had synergistic effect with blocking of PD-1 andCTLA-4.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation.

The invention claimed is:
 1. An isolated monoclonal antibody which bindsto human T-cell immunoglobulin and ITIM domain (TIGIT), or an antibodyfragment thereof comprising at least the antigen binding portion of themonoclonal antibody, wherein the isolated antibody or antibody fragmentis selected from the group consisting of: (i) an isolated antibody orantibody fragment thereof comprising six complementarity determiningregions (CDRs), wherein the heavy chain (HC) CDR1 comprises the sequenceGYTFTSYGIS (SEQ ID NO:1) or TSYGIS (SEQ ID NO:11); the HC CDR2 comprisesthe sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2); the HC CDR3 comprises thesequence: KGPYYTKNEDY (SEQ ID NO:3); the light chain (LC) CDR1 comprisesthe sequence: RASEHIYYSLA (SEQ ID NO:4); the LC CDR2 comprises thesequence: NANSLED (SEQ ID NO:5); and the LC CDR3 comprises the sequence:KQAYDVPRT (SEQ ID NO: 6); and (ii) an isolated antibody or antibodyfragment thereof comprising six CDRs, wherein the HC CDR1 comprises thesequence IYCIH (SEQ ID NO:12); the HC CDR2 comprises the sequence:EISPSNGRTIYNEKFKN (SEQ ID NO:13); the HC CDR3 comprises the sequence:SDGYDGYYFDY (SEQ ID NO:14); LC CDR1 comprises the sequence: RASQEISGYLN(SEQ ID NO:15); the LC CDR2 comprises the sequence: AASTLDS (SEQ IDNO:16); and the LC CDR3 comprises the sequence: LQYASYPRT (SEQ IDNO:17).
 2. The isolated monoclonal antibody or the antibody fragmentaccording to claim 1, comprising a heavy chain and a light chain,wherein the heavy chain comprises SEQ ID NO:7 and the light chaincomprises SEQ ID NO:8.
 3. The isolated monoclonal antibody or theantibody fragment according to claim 1, comprising a heavy chain and alight chain, wherein the heavy chain comprises SEQ ID NO:18 and thelight chain comprises SEQ ID NO:19.
 4. The isolated monoclonal antibodyof claim 1 wherein the antibody is selected from the group consistingof: a bispecific antibody, a humanized antibody, and an antibodyconjugate.
 5. The isolated monoclonal antibody of claim 4, wherein thebispecific antibody comprises two sets of CDR sequences, wherein: oneCDR set comprises an HC CDR1 having the sequence GYTFTSYGIS (SEQ IDNO:1) or TSYGIS (SEQ ID NO: 11); an HC CDR2 having the sequence:EIYPRSGNTYYNEKFKG (SEQ ID NO:2); an HC CDR3 having the sequence:KGPYYTKNEDY (SEQ ID NO:3); an LC CDR1 having the sequence: RASEHIYYSLA(SEQ ID NO:4); an LC CDR2 having the sequence: NANSLED (SEQ ID NO:5);and an LC CDR3 having the sequence: KQAYDVPRT (SEQ ID NO:6); and asecond set comprises an HC CDR1 having the sequence: IYCIH (SEQ IDNO:12), an HC CDR2 having the sequence: EISPSNGRTIYNEKFKN (SEC IDNO:13), an HC CDR3 having the sequence: SDGYDGYYFDY (SEC ID NO:14), anLC CDR1 having the sequence: RASQEISGYLN (SEC ID NO: 15), an LCCDR2having the sequence: AASTLDS (SEQ ID NO: 16), and an LC CDR3 having thesequence: LQYASYPRT (SEC ID NO:17).
 6. The isolated monoclonal antibodyaccording to claim 1 capable of inhibiting binding of TIGIT to at leastone ligand selected from the group consisting of PVR (CD155), PVRL2(CD112), PVRL3 (CD113), and any combination thereof.
 7. The monoclonalantibody according to claim 1 attached to a cytotoxic moiety, aradioactive moiety, or an identifiable moiety.
 8. A pharmaceuticalcomposition comprising as an active ingredient, at least one isolatedantibody or fragment thereof according to claim 1, and a pharmaceuticalacceptable excipient, diluent, salt, or carrier.
 9. A method of treatingcancer, comprising administering to a subject in need thereof, apharmaceutical composition according to claim
 8. 10. The method of claim9 further comprising administering an additional anti-cancer therapyselected from surgery, chemotherapy, radiotherapy, and immunotherapy.11. The method of claim 10, wherein the immunotherapy comprisingadministering an antibody that binds an immune checkpoint moleculeselected from the group consisting of PD-1, CTLA-4, PDL-1, CEACAM1,lymphocyte activation gene 3 (LAG3), CD137, OX40 (also referred to asCD134), killer cell immunoglobulin-like receptors (KIR), and anycombination thereof.
 12. The method of claim 10 wherein theimmunotherapy comprises administration of an antibody that inhibitsepidermal growth factor receptor (EGFR).
 13. A kit for measuring theexpression of TIGIT in biological sample comprising at least oneantibody or antibody fragment according to claim 1.